Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications and documents is incorporated by reference herein. The Sequence Listing is provided at the end of the Specification and before the claims.
Staphylococcus aureus (S. aureus) is an important pathogen in humans which is now under increasing risk of developing antibiotic resistance to currently available therapeutics. Consequently, there is a pressing need to identify new types of antibiotic agents effective against these drug resistant bacterial strains. The phenomenon of ‘bacterial interference’ may provide as yet unexplored avenues for the design of these new therapeutics. Bacterial interference refers to the ability of one organism to disrupt the biological functions of another. Until recently this survival process was thought to occur solely through a growth inhibition mechanism (Ji et al. (1997) Science 276, 2027-30), however a novel type of bacterial interference in S. aureus has been described which involves the inhibition of the so-called agr response (Novick et al. (1995) Mol. Gen. Genet. 248, 446-58; Morfeldt et al. (1995) EMBO J. 14:4569-4577). This process is mediated by short secreted peptides containing a putative thiololactone ring structure. Chemical synthesis confirms that the native Agr peptides contain a thiololactone moiety, and that this structure is absolutely necessary for full biological activity. In addition, structure-activity studies are described by the present invention which offer insights into the nature of the agr activation and inhibition mechanisms.
Accessory genes allow bacteria to survive and multiply in plant or animal hosts. In S. aureus these virulence factors (cytotoxins and tissue-degrading enzymes) are under the control of the agr locus which contains two divergent promoters, P2 and P3. The RNA transcript from the P3 promoter is responsible for the upregulation of secreted virulence factors as well as the downregulation of surface proteins, the agr response (Novick et al. (1993) EMBO J. 12, 3967-75; Morfeldt et al. (1995) EMBO J. 14:4569-4577). There are four genes, agrA-D, in the P2 operon which code for the cytosolic, transmembrane and extracellular components of a density-sensing/autoinduction circuit (Novick et al. (1995) supra). The product of the agrD gene is a pro-peptide which is processed and secreted through AgrB, an integral membrane protein. The active AgrD peptide is then thought to bind to the transmembrane receptor by the agrC gene. Binding of the AgrD peptide triggers a standard two-component signal transduction pathway in which the AgrC receptor becomes autophosphorylated on a histidine residue leading to subsequent trans-phosphorylation of the AgrA gene product. Phosphorylated AgrA then activates transcription from the P2 and P3 agr promoters (Novick et al. (1995) supra).
S. aureus strains can be divided into a least three groups (Ji et al. (1997) supra), each of whose secreted AgrD peptide can activate the agr response within the same group and inhibit the agr response in strains belonging to the other groups. It is the latter effect that constitutes a novel form of bacterial interference (Ji et al. (1997) supra). The AgrD autoinducing peptides, generated following processing and secretion through AgrB, consist of seven to nine residues. Interestingly, the sequences are highly variable among the groups, although all contain a conserved cysteine residue 5 amino acids from the C-terminus. Mass spectrometric analysis of AgrD peptides isolated from culture supernatants indicated a mass discrepancy of −18 Da compared to the predicted masses based on the peptide sequences (Ji et al. (1995) Proc. Natl. Acad. Sci. USA 92, 12055-9). This observation combined with the presence of the conserved cysteine residue in AgrD peptides, has led to the suggestion that these secreted peptides contain an intramolecular thiol ester linkage between the cysteine sulfhydryl group and the carboxy-terminus (Ji et al. (1997) supra). Consistent with this thiololactone structure, the addition of hydroxylamine to a purified AgrD peptide was observed to abolish its biological activity (Ji et al. (1997) supra).
The inability to isolate significant quantities of secreted AgrD peptides means that very little is known about the biochemistry of the AgrD/AgrC interaction. For example, the potency of the AgrD peptide in either activating (within S. aureus strains of the same group) or inhibiting (in S. aureus strains from other groups) the agr response is unknown. Equally, it is essential to determine whether the putative thiololactone structure within the AgrD peptides is required for activation of the agr response, inhibition of the agr response or both. The present disclosure provides such elucidation. The study detailed herein confirms the presence of the thiololactone moiety within the AgrD peptides through total chemical synthesis. Having demonstrated synthetic access to the system, more rigorous biochemical and structure-activity studies on the AgrD/AgrC interaction are addressed. The present disclosure further delineates that elimination of the thiol ester component of the cyclic ring structure can destroy activity activating the agr response while preserving (and enhancing) inhibitory activity.